Mutations play both a positive and negative central role in an living organisms. Single base substitutions are the simplest class of mutations, yet they can have profound biological consequences. They can act as a driving force governing evolution. There are also numerous examples of cancers that can result from an alteration of just one base pair in the human genome. The same is true for inherited diseases such as sickle cell anemia and Lesch-Nyhan syndrome. It is well known since the earliest mutagenesis studies following the elucidation of the structure of DNA, that mutations occur in a nonrandom fashion along the genome. Certain sites along DNA, referred to as "hot spots", exhibit much higher than average mutation frequencies, while other sites, "cold spots", mutate at a significantly diminished rate. The focal point of this grant proposal is to investigate base substitution hot and cold spots at a molecular level. It is well known that proximal and even distal base sequences can strongly affect base substitutions at a given DNA locus. We have proposed that nearest-neighbor base stacking forces modulate the fidelity of nucleotide insertion of DNA polymerase and the efficiency of elongating DNA containing a single base mismatch. It has also been suggested that the relative stability of the DNA (i.e., the ratio of A-T/G-C base pairs) can profoundly affect the efficiency of error correction, by proofreading exonucleases, at the replication fork. We are utilizing a polyacrylamide gel assay to test models of polymerase fidelity as a function of base context. Each fidelity component: nucleotide insertion, extension and/or excision can be measured independently. We will determine whether interesting correlations we had earlier observed between nearest neighbor nucleotides and enzyme discrimination mechanisms can be supported by obtaining additional independent data. In addition to base substitutions involving normal base mispairs, there are biologically significant lesions that can affect the templating properties of DNA. The loss of a base results in a noninstructional (abasic) lesion, while base alkyations can alter base pairing specificities. Abasic sites and alkyated bases are known to be highly deleterious lesions, causing replication blockage, mutagenesis, and carcinogenesis. We intend to use the gel fidelity assay to analyze the effects of base context on insertion, extension and proofreading at selected lesion sites on DNA. Finally, allele selective amplification using the polymerase chain reaction has become an important method to identify mutations of human origin in cloned DNA. Our experiments on the effects of base context on extension past a lesion will have practical application in the design and analysis of these experiments.